Pig species identification in meatballs using polymerase chain reaction-restriction fragment length polymorphism for
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International Food Research Journal 19 (3): 901-906 (2012)
Pig species identification in meatballs using polymerase chain
reaction- restriction fragment length polymorphism for
Halal authentication
1,3*
Erwanto, Y., 1 Abidin, M.Z., 2,3 Sismindari and 2,3Rohman, A.
1
Division of Animal Products Technology, Faculty of Animal Science, Gadjah Mada
University, Jl. Fauna No. 3, Bulaksumur, Yogyakarta 55281, Indonesia
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gadjah Mada
University, Jl. Kaliurang Km 4,5, Sekip, Yogyakarta 55281, Indonesia
3
Halal Products Research Centre, Gadjah Mada University, Jl. Kaliurang Km 4,
Sekip, Yogyakarta 55281, Indonesia
Abstract: The detailed information on the chemical and nutritional content is essential for consumers in choosing
meat-derived food products. For moslem communities, it is prohibited to consume pork-contained or other pig
derivatives foods. Unfortunately, meat adulteration by means of mixing beef and chicken with pork or other pig
derivatives frequently occurs in the market. This habits cause difficult identification of beef and chicken that
are free from pork and other derivatives products. Genomic DNA of pig, bovine, and chicken were isolated and
subjected to PCR amplification targeting the mitochondrial cytochrome b gene. Pig species differentiation was
determined by digestion of 359 bp amplified product obtained with BseDI restriction enzymes, which generated
pig species electrophoresis pattern. PCR-Restriction Fragment Length Polymorphism (RFLP) revealed the
presence of pork in meatball product which can be distinguished among bovine, chicken, and pig samples.
Pig mitochondrial cytochrome DNA gene was cleaved into 228 bp and 131 bp fragments but the bovine, and
chicken cytochrome b gene were not digested by BseDI enzyme. PCR-RFLP technique using BseDI restriction
enzymes is reliable for the detection of pork in meatball for the Halal authentication.
Key words: Pig species, Identification, PCR-RFLP, Halal authentication
Introduction of the value(s) obtained with those previously
documented for authentic material of the same type.
Indonesian traditional meatballs or known as This approach is often time-consuming and therefore
“bakso” is one of the comminuted meat products and expensive; therefore, some analytical methods
gains the popularity among all classes of Indonesian offering fast and reliable results are continuously
society. The products are served in hot soup with developed by some researchers (Downey, 1998). One
other stuffs such as tofu, noodle, cabbage and chili of them is DNA-based methods.
or tomato sauce. Meat used to make bakso originally Many various methods based on DNA techniques
comes from beef, but nowadays some others such as have developed such as multiplex PCR assay
chicken, fish, and pork are also commonly used in (Matsunaga et al., 1999) and PCR-based finger
some meatball products (Purnomo and Rahardiyan, printing (Saez et al., 2004). Colgan et al. (2001)
2008). The wide variety of meatball products availabe analyzed meat bone meal using real time PCR to
on the market in Indonesia seems favourable but leads investigate the meat source origin and to verify the
to several fears for Muslim community, because the quantity of meat in DNA mixture complex. Lopes-
presence of pork in meatball products are prohibited andreo et al. (2005) also studied meat species
to be consumed (Rohman et al., 2011). This is an identification using the same methods. Similarly,
important challenge for the people in charge of the identification of the added por or porcine in a mixture
official control of food which have an obligation to of meat products can be carried out based on the
to verify the species of meat ingridients that are not identification of porcine DNA. Therefore, the aim of
always easily identifiable. this study was to apply the PCR-RFLP technology as
The strategies used to detect the adulterated a tool for meat species identification on samples of
products have traditionally relied on wet chemistry the Indonesian meatballs.
to determine the amount of a marker compound or
compounds in a test material followed by a comparison
*Corresponding author.
Email: erwantougm@gmail.com (Y. Erwanto) © All Rights Reserved
Tel: +62-274-513363, Fax : +62-274-521578
902 Yuny, E., Mohammad, Z. A., Sismindari and Rohman, A.
Materials and Methods West Sussex, UK), and 20 pmol of each primer.
Amplification was performed with a thermal cycler
Sample preparation and DNA extraction according to the following PCR step-cycle program:
Authentic muscle samples of beef, pork and pre-denaturation of 94°C for 2 min to completely
chicken were obtained from the traditional market denature the DNA template, followed by 35 cycles of
in Yogyakarta, Indonesia. Meatball was prepared denaturation at 95°C for 36 s, annealing at 51°C for
in laboratory scale with separate equipment to 73 s, and extension at 72°C for 84 s. Final extension at
prevent cross contamination. Meatball samples were 72°C for 3 min followed the final cycle for complete
prepared by mixing pork with beef or chicken at a synthesis of elongated DNA molecules. Two
final concentration of pork at 0; 1.0; 2.5; 5.0; 10.0 microlitres of PCR products were electrophoresed at
and 25.0 % (w/w). constant voltage (50V) on 2% agarose gel (Promega,
DNA was extracted from meatball samples using Madison, USA) for about an hour in 1x TBE buffer,
the High Pure PCR Template protocol for animal pH 8.0 and stained by ethidium bromide. A-100 bp
tissue provided with the High Pure PCR Template DNA ladder (Promega, Madison, USA) was used
Kit (Roche, Germany). Approximately 50-100 mg of as size reference. The gel photo was taken using the
meatballs was blended using a commercial blender Syngene gel documentation system.
and placed in a 1,5 ml microcentrifuge tube. A-100 µl
of tissue buffer and 40 µl Proteinase K were added Restriction fragment length polymorphism
and mixed by vortexing. The mixture was incubated Two units/µl of RE BseDI (Fermentas) were
at 55°C in a water bath overnight to disperse the applied to 10 µl of amplified DNA in a final volume
sample until the tissue was completely lysed. The of 20 µl digestion mixture [containing 1x reaction
samples were then added with 200 µl binding buffer buffer (10 mM Tris-HCl, 100 mM KCl, 1 mM EDTA,
and incubated at 70°C for 10 min. The mixture was 0,2 mg/ml BSA, 1 mM DTT and 50% glycerol)] and
mixed b vortexing for seconds, added with 100 µl were incubated at 55°C for 3 h for optimal result. A-5
isopropanol, mixed vigorously and placed high filter µl of the digested samples were electrophoresed at
tubes. The samples was subsequently poured in the constant voltage (50 V) on 2% agarose gel (Promega,
collection tube, placed in table top centrifuge, and Madison, USA) for about an hour in 1x TBE buffer,
spun at 8,000 g for 1 min. The flow-through and pH 8.0 and stained by ethidium bromide. A-100 bp
collection tube were discarded and the High Filter (Promega, Madison, USA) was used as size reference.
Tube was placed in a new 2 ml collection tube. A-500 The gel photo was taken using the Syngene gel
µl of wash buffer was added and spun at 8,000 g for documentation system.
1 min. The flow-through and collection tube were
discarded and the High Filter Tube was placed in Results and Discussion
another 2 ml collection tube. The high filter tube
was dried by centrifugation for 10 seconds, and the PCR based amplification was carried out based
supernatant flow-through was discarded. The High on the sequence of the mitochondrial cytochrome
Filter Tube was placed in a clean 1.5 ml micro b of the products. For restriction fragment length
centrifuge tube. A-200 µl of pre-warmed elution polymorphism was carried out by digesting the PCR
buffer was added and spun at 8,000 g for 1 min to products using BseD I enzymes. Genomic DNA
elute. The DNA solution was stored at 4 °C. isolation from the meatball can be extracted with this
kit, but it is ascribed to the fact that thermal strongly
PCR amplification of a conserved Cytochrome 2b of accelerates DNA degradation from the meatball
Mitochondrial gene fragment samples (Figure 1). The data was in comfort with
The set of primers used for amplification the finding of Arslan et al. (2006) and Tanabe et
consisted of Cyt b-FW and Cyt b-REV oligonucleotides al. (2007) who reported that heating of the samples
as follows: CYT b FW 5’-CCA TCC AAC ATC TCA by various treatment did not significantly affect the
GCA TGA TGA AA-3’, CYTb REV 5’-GCC CCT DNA and it was able to detect. Matsunaga et al.
CAG AAT GAT ATT TGT CCT CA-3’. Amplification (1999) has also studied of DNA isolation in meat
of the mt cyt b gene was performed in a final volume which was processed with high temperature around
of 25 µl containing 250 ng of extracted DNA, mega- 100 and 120°C for 30 min of various meat flesh such
mix royal (optimized mixture of Taq polymerase, as cattle, goat, chicken, sheep, horse and pig, while
anti-Taq polymerase monoclonal antibodies in 2 X Tanabe et al. (2007) provided similar data of pork
reaction buffer (6 mM MgCl2 with 400 µM dNTPs, at various cooked. According to Martinez and Yman
stabilizer and blue loading dye) (Microzone Ltd, (1998) and Saez et al. (2004), the heat treatments
International Food Research Journal 19(3): 901-906
Pig species identification in meatballs for Halal authentication 903
which mainly affected the quality DNA can cause the
DNA degradation into small size fragment.
Figure 2. PCR products of cytochrome b gene fragments
359 bp long of samples from different meatballs
Figure 1. Total genomic DNA extracted from beef-pork product separated by 2% high-resolution agarose gel
meatball and chicken-pork meatball. (A) M: marker 100 electrophoresis. PCR amplification using cyt b universal
bp DNA ladder (Invitrogen), 1: pork (100%), 2: (beef 75% primer. (A) M: marker 100 bp DNA ladder (Invitrogen),
: pork 25%) 3: (Beef 90% : Pork 10%), 4: (Beef 95% : 1: pork (100%), 2: (beef 75% : pork 25%) 3: (Beef 90% :
Pork 5%)5: (Beef 97% : Pork 3%), 6: (Beef 99% : Pork Pork 10%), 4: (Beef 95% : Pork 5%)5: (Beef 97% : Pork
1%), 7: (Beef 100 %). (B): M: marker 100 bp DNA ladder 3%), 6: (Beef 99% : Pork 1%), 7: (Beef 100 %). (B): M:
(Invitrogen), 1: pork (100%), 2: (chicken 75% : pork 25%) marker 100 bp DNA ladder (Invitrogen), 1: pork (100%),
3: (chicken 90% : Pork 10%), 4: (Chicken 95% : Pork 2: (chicken 75% : pork 25%) 3: (chicken 90% : Pork 10%),
5%)5: (Chicken 97% : Pork 3%), 6: (Chicken 99% : Pork 4: (Chicken 95% : Pork 5%)5: (Chicken 97% : Pork 3%),
1%), 7: (Beef 100 %). 6: (Chicken 99% : Pork 1%), 7: (Beef 100 %).
International Food Research Journal 19(3): 901-906904 Yuny, E., Mohammad, Z. A., Sismindari and Rohman, A.
Genomic DNA was applied as a template for the
PCR amplification using universal primers. Gene of
cytochrome b was selected for the PCR amplification
and resulted a DNA fragment of approximately 359 bp
(Figure 2). This result indicated that isolated DNA of
mixture meatball was enough for PCR amplification.
The same result of PCR amplification has also been
reported previously (Kocher et al., 1989; Aida et
al., 2005; Erwanto et al., 2011). The selection of
target gene and primers affecting sensitivity and
specification of method for detection. PCR method
Figure 3. Sequences of nucleotide cytochrome b of Sus scrofa (pig) restriction site by BseDI using CLC
Sequencing Software and universal primer position in the fragment of cytochrome b gene (→ dan ←).
was very sensitive when primer target represent a
gene multicopy of like gene mitochondrial. This
research used the area mitochondrial DNA of the
cytochrome b as target for detection of porcine.
The PCR reaction allowed fragments of the
expected length to be obtained in all meatball samples
either beef or chicken mixed with pork, although with
various efficiencies. The mitochondrial cytochrome
b gene was selected in this study as template for DNA
amplification, because it has an acceptable length and
an adequate grade of mutation and there are numerous
sequences available in the DNA bank databases
(Kocher et al., 1989). The mitochondrial primers Cyt
b-FW and Cyt b-REV was able to amplify a conserved
359 bp region of the cytochrome b gene of all animal
studied, namely chicken, beef and pork.
Sequence DNA of cytochrome b gene of cattle,
goat, chicken and pig obtained from database of
NCBI was further employed for sequence alignment
using software of CLC sequencer. The similarity of
the mitochondrial cytochrome b gene among beef,
mutton, chicken and pork was 86.64%. As a result
of the preliminary CLC sequencer software analysis
for the detection of specific restriction sites on pig
sequence, a site recognized by BseDI enzyme was
cleaved into two fragments, namely 131 bp and 228
bp (Figure 3). Based on RFLP pattern using CLC
sequencer, BseDI was applicable to differentiate or
identify among four species.
The digestion of PCR products resulted the
different fragment sizes, it was 131 and 228 bp at PCR
product of porcine. Basically, PCR product of mutton
could also be digested, but DNA length size was very
short (approximately 5-20 bp), consequently, it could
not be seen at 2% agarose gel (Figure 4). A clear band
with a length between 100 and 150 bp was observed
and thus referable to the 131 bp fragment, as shown
in Figure 4 (lane 1). In the same lane, a thicker band
can be traced back to the 228 bp fragment.
The data obtained suggests that compared with
BsaJI endonuclease profiles, the DNA restriction
patterns obtained after digestion of the amplicons
with BseDI enzymes consisted of same patterns.
International Food Research Journal 19(3): 901-906Pig species identification in meatballs for Halal authentication 905
The difference between BsaJI and BseDI restriction
enzyme is the incubation time for the digestion. BseDI
needed 3 h for digestion, while BsaJI enzyme needed
more than 12 h (Aida et al., 2005).
PCR amplification of cytochrome b gene followed
by digestion by BseDI restriction enzymes was a
powerful technique for the identification of pork or
other pig derivative products contamination, due to
its simplicity and sensitivity. The cytochrome b gene
alignment using CLC sequencer software showed
that pig intra species have the same restriction sites
and their homology was 98.2%.
Conclusions
Our results allow us to conclude that PCR-RFLP
of the mitochondrial Cytochrome b gene is a suitable
alternative technique that can be applied to the
detection of pig species present in the commercialized
food products such as meatballs.
Acknowledgement
This research was financially supported by
grants from Riset Unggulan Strategis Nasional
LPPM Universitas Gadjah Mada (Grant number
LPPM-UGM/1309/2009). The authors also deeply
thanks to Dr. Widodo for the critical reading of this
manuscript.
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